人鼻咽癌细胞基因组中瘤基因Ha－ras－1的Dnase Ⅰ超敏感位点的初步研究

ＤＮａｓｅⅠ超敏感位点的研究能够发现潜在的调控基因转录活化的位点,比较正常人外周血有核细胞,淋巴瘤细胞株Ｐ３ＨＲ１和人鼻咽癌低分化磷癌细胞株ＨＯｎＥ１和ＨＮＥ２中Ｈａ－ｒａｓ－１瘤基因的ＤＮａｓｅⅠ超敏感位点发现,只有ＨＯＮＥ１和ＨＮＥ２细胞基因组中存在一个ＤＮａｓｅⅠ超敏感位点,位于第一个外显子上游０．３７ｋｂ处,上述结果提示正常白细胞和Ｐ３ＨＲ１细胞中Ｈａ－ｒａｓ－１基因处于失活状态,而在鼻咽癌细胞基因组中则处于活化状态,它的活化可能与０．３７ｋｂ处的ＤＮＡ序列有密切的关系。     

人染色体脆性位点部位的显微光谱学研究 Study of Microspectroscopy for the Position of the Fragile Sites in Human Chromosome

采用显微分光光度法,对染色体脆性位点的部位进行了显微光谱学研究。实验证明,带有脆点的染色体其DNA含量大多数趋向减少,少数略有增加,推测染色体脆性部位的产生是由于染色质DNA在高度凝缩形成中期染色体过程中超旋结构改变的结果。 The position of fragile sites in human chromosome was studied by means of the microspectroscopy. The results show that the amount DNA in chromosome with fragile sites decreases in most condition. We can suppose that the fragile sites of chromosome is caused by the superhelix structure changes of chromosome DNA during the formation of metaphase chromosome which is formed in high condensation.     

Chemically modified cardiac Na+ channels and their sensitivity to antiarrhythmics: Is there a hidden drug receptor?

Elementary Na⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes. In analyzing the sensitivity of chemically modified Na⁺ channels to several class 1 antiarrhythmic drugs, the hypothesis was tested that removal of Na⁺ inactivation may be accompanied by a distinct responsiveness to these drugs, open channel blockade.Iodate-modified and trypsin-modified cardiac Na⁺ channels are noninactivating but strikingly differ from each other by their open state kinetics, a O₁–O₂ reaction (_open(1) 1.4±0.3 msec; _open(2) 5.4±1.1 msec; at –40 mV) in the former and a single open state (_{open 3.0±0.5} msec; at –40 mV) in the latter. Lidocaine (150 mol/liter) like propafenone (10 mol/liter), diprafenone (10 mol/liter) and quinidine (20 mol/liter) in cytoplasmic concentrations effective to depress NP _o significantly can interact with both types of noninactivating Na⁺ channels to reduce the dwell time in the conducting configuration. lodate-modified Na⁺ channels became drug sensitive during the O₂ state. At –40 mV, for example, lidocaine reduced _open(2) to 62±5% of the control without detectable changes in _open(1). No evidence could be obtained that these inhibitory molecules would flicker-block the open Na⁺ pore. Drug-induced shortening of the open state, thus, is indicative for a distinct mode of drug action, namely interference with the gating process. Lidocaine proved less effective to reduce _open(2) when compared with the action of diprafenone. Both drugs apparently interacted with individual association rate constants, a_lidocaine was 0.64×10⁶ mol^–1 sec^–1 and a_diprafenone 13.6×10⁶ mol^–1 sec^–1. Trypsin-modified Na⁺ channels also appear capable of discriminating among these antiarrhythmics, the ratio a_diprafenone/a_lidocaine even exceeded the value in iodate-modified Na⁺ channels. Obviously, this antiarrhythmic drug interaction with chemically modified Na⁺ channels is receptor mediated: drug occupation of such a hypothetical hidden receptor that is not available in normal Na⁺ channels may facilitate the exit from the open state.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-4), Bonn.     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

Chronic Nicotine Administration Differentially Affects Neurotransmitter Release from Rat Striatal Slices

Abstract: The objective of these experiments was to determine whether the chronic administration of nicotine, at a dose regimen that increases the density of nicotine binding sites, alters the nicotine-induced release of [³H]dopamine ([³H]DA), [³H]norepinephrine ([³H]NE), [³H]serotonin ([³H]5-HT), or [³H]acetylcholine ([³H]ACh) from rat striatal slices. For these experiments, rats received subcutaneous injections of either saline or nicotine bitartrate [1.76 mg (3.6 µmol)/kg, dissolved in saline] twice daily for 10 days, and neurotransmitter release was measured following preloading of the tissues with [³H]DA, [³H]NE, [³H]5-HT, or [³H]choline. Chronic nicotine administration did not affect the accumulation of tritium by striatal slices, the basal release of radioactivity, or the 25 mM KCl-evoked release of neurotransmitter. Superfusion of striatal slices with 1, 10, and 100 µM nicotine increased [³H]DA release in a concentration-dependent manner, and release from slices from nicotine-injected animals was significantly (p < 0.05) greater than release from saline-injected controls; release from the former increased to 132, 191, and 172% of release from the controls following superfusion with 1, 10, and 100 µM nicotine, respectively. Similarly, [³H]5-HT release increased in a concentration-related manner following superfusion with nicotine, and release from slices from nicotine-injected rats was significantly (p < 0.05) greater than that from controls. [³H]5-HT release from slices from nicotine-injected rats evoked by superfusion with 1 and 10 µM nicotine increased to 453 and 217%, respectively, of release from slices from saline-injected animals. The nicotine-induced release of [³H]NE from striatal slices was also concentration dependent but was unaffected by chronic nicotine administration. [³H]ACh release from striatal slices could not be detected when samples were superfused with nicotine but was measurable when tissues were incubated with nicotine. The release of [³H]ACh from slices from nicotine-injected rats was significantly (p < 0.05) less than release from controls and decreased to 36, 83, and 77% of control values following incubation with 1, 10, or 100 µM nicotine, respectively. This decreased [³H]ACh release could not be attributed to methodological differences because slices from nicotine-injected rats incubated with nicotine exhibited an increased [³H]DA release, similar to results from superfusion studies. In addition, it is unlikely that the decreased release of [³H]ACh from striatal slices from nicotine-injected rats was secondary to increased DA release because [³H]ACh release from slices from hippocampus, which is not tonically inhibited by DA, also decreased significantly (p < 0.05) in response to nicotine; hippocampal slices from nicotine-injected rats incubated with 1 and 10 µM nicotine decreased to 42 and 70%, respectively, of release from slices from saline-injected animals. Results indicate that the chronic administration of nicotine increases the ability of nicotine to induce the release of [³H]DA and [³H]5-HT and decreases the ability of nicotine to evoke the release of [³H]ACh but does not alter the nicotine-induced release of [³H]NE from brain slices.     

Temperature Sensitivity of Agonist High-Affinity Binding Sites of Solubilized and Reconstituted D1 Dopamine Receptors

Abstract: Solubilization of rat striatal membranes with sodium cholate, followed by reconstitution into phospholipid vesicles, leads to a 6.5-fold increase in the agonist high-affinity binding sites of the D₁ dopamine receptor. These high-affinity binding sites display differential sensitivity toward temperature. When reconstituted receptors were preincubated for 1 h at 0–4°C (on ice) or at 22°C (room temperature) followed by radioligand binding assays with dopamine, neither the high-affinity values of the receptor for dopamine nor the percent receptors in the high-affinity state (31–39%) were changed from control reconstituted receptors, which were not subject to any preincubations. At 30°C, there was a partial loss in the number of high-affinity D₁ receptors with only 25% of the total receptor population in the high-affinity state; there was no change in the affinity values of the high-affinity binding sites. At 37°C, there was a 40% loss in total number of D₁ receptor binding sites. All the high-affinity binding sites were lost and the remaining 60% of binding activity represented the low-affinity binding state of the receptor. These results indicate that the high-affinity binding sites of the reconstituted D₁ dopamine receptors are uniquely sensitive to higher temperatures.     

RFLP mapping of partially sequenced leaf cDNA clones in maize

We report here the results of mapping a set of 92 leaf cDNA clones in maize. The ends of each of these cDNA clones have previously been partially sequenced, and the sequence comparison has revealed the putative function for 28 clones. It is expected that the RFLP map developed using these expressed sequence tags will be of great importance for future maize genome analysis, such as for PCR-based gene mapping or gene function identification.Contribution from the Missouri Agricultural Experiment Station. Journal Series N. 12,019.     

Morphology and aestivation behaviour in some Madagascan acavid land snails

Nine species of Madagascan acavid land snails were compared in a phylogenetic context. The two most plesiomorphic, Clauator johnsoni and C. moreleti, differ from the others by their high-spired shells, short tentacles, short tails, long necks, and crawling mode of hitching the shell along the ground. In the seven more apomorphic species, the crawling mode is smooth, with the shell resting on the tail, and the relative lengths of tail and shell correlate significantly. Among these seven species, three pairs of closest relatives (Helicophanta petiti and H. uesicalis, H. farafanga and H. souuerbiana, Ampeltta decaryi and A. julii) show evidence of phylogenetic constraints on ranked shell size. Aestivation site (as tentatively inferred from rare data) does not correlate with shell shape or size: burrowers have H/D = 2.7 to 0.6 and D = 70 to 25 mm; arboreals have H/D = 0.8 to 0.5 and D = 70 to 30 mm; the species with both the highest spire and the smallest diameter (C. moreleti) is neither a burrower or an arboreal, but stays on the ground surface. Inferred aestivation sites are randomly distributed phylogenetically. Climate shows no correlation, except that the arborcals are only from humid to wet regimes. Uniform shell colouration occurs only in burrowers (C. johnsoni, H. petiti, H. uestcalis), but disruptive shell colouration occurs in all others, including burrowers (H. farafanga, A. decaryt), ground-surface aestivators (C. moreleti), arboreals (H. souuerbiana, A. julii), and semi-arboreals (Ampefita subfunebris). Among all nine species, burrowers have significantly thicker shells (than their close relatives of similar size), wider bodies, and longer snouts than non-burrowers (H. souuerbiana is exceptional in being arboreal despite its huge size and in having the broad foot and snout of a burrower). Thus, although there is some evidence for phylogenetic constraints, natural selection for aestivation and crawling behaviours seems to have dominated the evolution of external body morphology and of shell thickness (but not shell size and shape) in these snails.     

Binding of malate dehydrogenase and NADH channelling to complex I

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.